Precipitation of IgG (recombinant antibody) with a hydrophobic polymer (polyethylene glycol=PEG) is an alternative method to the conventional protein A affinity chromatography for purification of monoclonal antibodies. Protein precipitation with PEG was established by Polson (Polson et al. 1964). Atha and Ingham (Atha and Ingham 1981) showed that the concentration of precipitated protein increases with increasing PEG concentration and that the precipitation is also influenced by PEG size and pH value (Polson et al. 1964). It is assumed that the precipitation mechanism with polyethylene glycol is a volume exclusion reaction (Mahadevan and Hall 1990). PEG can be used to precipitate large proteins (e.g., host cell proteins (HCP)) as well as DNA. However, separation of IgG from DNA (as well as other larger proteins) with PEG as differences in molecular mass by a factor of about two is required to approach complete (e.g., 100%) separation (Lis and Schleif 1975). To obtain the required DNA and HCP clearance, PEG precipitation may be combined with another precipitation approach. Caprylic acid precipitation is a common way to separate venom out of horse plasma (Rojas et al. 1994). In several publications the precipitation parameter for caprylic acid precipitation are described (McKinney and Parkinson 1987; Mohanty and Elazhary 1989; Russo et al. 1983). Caprylic acid precipitation was combined with different purification methods like ammonium sulphate precipitation to purify human immunoglobulins (Perosa et al. 1990) or with ion-exchange chromatography to separate equine antivenom (Raweerith and Ratanabanangkoon 2003). Also, a CA precipitation of process-derived impurities out of CHO cell culture supernatant combined with an additional cation-exchange chromatographic step has also been performed (Wang et al. 2009).
Caprylic acid has also been used to precipitate non-immunoglobulin proteins from human plasma. The crude IgG present in the supernatant (which contained 26-29% of the total protein in terms of absorbance units or 78-87% of IgG by weight) was fractionated on DEAE-cellulose, to yield pure IgG as shown by disc electrophoresis, Immunoelectrophoresis and gel filtration. Pure IgG was free of plasmin and plasminogen and did not exhibit any fragmentation or aggregation during storage for periods up to 4 weeks at 40° C., and its anticomplementary activity was low. Antibodies to viral agents were recovered unchanged. (Habeeb, et al. Preparation of Human Immunoglobulin by Caprylic Acid Precipitation. Preparative Biochemistry, 14(1): 1-17 (1984)).
Parkkinen, et al. (Vax Sanguinis, 90: 97-104 (2006)) describe the isolation of pure, essentially polymer-free IgG from human Cohn fraction II+III by caprylic acid treatment to inactivate enveloped viruses and precipitate contaminating proteins and lipids, polyethylene glycol (PEG) precipitation of IgG, and final purification by anion-exchange chromatography. US 2007/0244305 A1 (Parkkinen, J.) described a similar process.
Bergmann-Leitner, et al. (Malaria J. 7: 129-139 (2008)) compares various immunuglobulin (Ig) procedures using immunized rabbit sera or malaria-exposed human sera as the Ig source. Procedures tested included enrichment of total Ig by caprylic acid depletion of serum proteins followed by ammonium sulfate precipitation (CA-AS). The CA-AS method was found to provide good yield without significant degradation. PEG precipitation was found to be optimal for isolating human Ig.
Vargas, et al. (Biotechnol. Prog., 28(4): 1005-1011 (2012)) describes the separation of human IgG from albumin as upper and lower phases by PEG precipitation. IgG present in the upper phase was then further isolated by caprylic acid precipitation and ion exchange chromatography.
U.S. Pat. No. 4,164,495 (Hansen, J.) describes methods for isolating immunoglobulins (Ig) using a polycondensed di or polyol, such as polyethylene glycol, in the presence of a mono or polyalkanoic acid having 4 to 12 carbon atoms, such as caprylic acid. Described therein is a method for isolating Ig from human blood plasma by first precipitating fibrinogen therefrom using PEG alone, isolating the precipitate comprising Ig, dissolving the same in sodium chloride solution, and admixing the solution with caprylic acid and PEG. The resulting precipitate is removed and the liquid phase precipitated with PEG alone to obtain “pure” Ig at a 60% yield.
U.S. Pat. No. 5,164,487 (Kothe, et al.) relates to methods for isolating “intravenously tolerable” Ig by treating a raw fraction containing IgG obtained by chromatography or Cohn processes and enriching the IgG using 0.4 to 1.5% octanoic acid (by volume) and chromatography (ion or cation exchanger or hydrophobic matrix). An “intravenously tolerable” preparation is described as one that may be administered to patients without side effects.
Steinbuch, et al. (Arch. Biochem. Biophys. 134: 279-284 (1969)) describes the use of caprylic acid to precipitate purified IgG (to 90% with caprylic acid/acetate buffer (pH 4.8)) and ion exchange cellulose and/or size exclusion chromatography (to remove IgA, ceruloplasmin and α1-acid-glycoprotein) from human serum. A similar procedure was used to isolate Ig from animal (horse, sheep, rabbit) serum.
McKinney (J. Immunol. Meth. 96: 271-8 (1987)) discloses a two-step procedure for isolating IgG from mammalian ascites fluid. The first step precipitates albumin and other non-IgG proteins with caprylic acid (octanoic acid) and the second precipitates IgG with ammonium sulfate (e.g., 80-90% recovery from rabbit serum).
U.S. Pat. No. 5,747,031 (Ruch, et al.) describes a method for isolating Ig from whey (e.g., made from hyperimmune milk) by co-precipitating lipids and non-Ig proteins simultaneously with a charged polymer (e.g., chitosan) and a fatty acid (e.g., caprylic acid). The resultant supernatant is then further treated by, for instance, diafiltration, to obtain isolated Ig (e.g., 66-79%).
WO 86/05099A1 (Steinbuch, et al.) relates to a process for isolating IgG4 from human plasma using a saturated fatty acid such as caprylic acid.
US 2002/177693AA (Lebing, et al.) describes a process for purifying antibodies from human plasma by preparing a composition comprising precipitated Ig, dissolving the Ig in a solution having a pH of 3.8 to 4.5, adding caprylate ions and increasing the pH to 5.0 to 5.2, and removing the precipitated proteins, lipids and caprylate by filtration. Caprylate is then added to the resultant solution, the temperature increased, and a filter aid introduced. The resultant precipitate is then removed by flow filtration. The solution is then passed through two anion exchange chromatography columns to produce Ig at >99% purity.
WO 2012/136172A1 (Segura Ruiz, et al.) describes a process for the production of injectable blood-derived protein materials by fractionating source material in a two-phase system including phenol, one phase being precipitated with caprylic acid and the other phase being processed by thermocoagulation.
Svendsen, et al. (Lab. Animal Sci. 45(1): 89-93 (1995)) compares the purification of antibodies from egg yolk by ammonium sulfate, PEG and caprylic acid, respectively. Ammonium sulfate was determined to provide the best purity and yield, and caprylic acid was found not to yield any purified antibodies.
Tscheliessnig, et al. (J. Chromatography, 1216: 7851-7864 (2009)) describes a two-step purification process for IgM (from hybridomas) using PEG and anion-exchange chromatography. Purity ranged from 46% to >95% with yields of 28-84%.
Knevelman, et al. (Biotechnol. Prog. 26: 697-705 (2010)) describes rapid PEG-based precipitation of IgG4 from cell culture media. Maximum yield and purity were determined to result from the use of between 10-18% PEG (w/v).
Kuczewski, et al. (Biopharm. Int., Supp., pp. 20-28 (March 2010)) described PEG-based precipitation of monoclonal antibodies from clarified cell culture media in yields of approximately 90% and reduced host cell protein (HCP) content by a factor of seven. Antibody was captured by cation exchange chromatography. Use of PEG-3350 was found to provide higher recovery but less HCP reduction. PEG-6000 was found to better reduce HCP but result in a lower yield. PEG-3350 at 14% (pH 8.5) was selected as the optimal condition.
Gibson, et al. (J. Pharm. Sci. 100(3): 1009-1021 (2011)) relates to the use of PEG with citrate, acetate, histidine or phosphate buffers across various pH values to test the solubility of IgG1 mAb produced by a CHO cell or murine cell line (each providing different glycosylation patterns to the mAb). These comparisons were made to identify strategies for use in high-throughput analysis of mAb preparations.
There is a need in the art for improved methods for isolating proteins such as antibodies from cell culture supernatants. This disclosure provides such improved methods.